ENZYME-SUBSTRATE INTERMEDIATE AT A SPECIFIC LYSINE RESIDUE IS REQUIRED FOR DEOXYHYPUSINE SYNTHESIS - THE ROLE OF LYS(329) IN HUMAN DEOXYHYPUSINE SYNTHASE

Deoxyhypusine synthase catalyzes the first step in the post-translatio nal synthesis of hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine] i n eukaryotic translation initiation factor 5A. We recently reported bi ochemical evidence for a covalent enzyme-substrate intermediate involv ing a specific lysine residue (Lys(329)) in human deoxyhypusine syntha se (Wolff, E. C., Polk, J. E., and Park, M. H. (1997) J. Biol. Chem. 2 72, 15865-15871), In an effort to evaluate the role of this enzyme-sub strate intermediate in catalysis, we carried out site-directed mutagen esis (Lys to Arg and/or Ala) of the conserved lysine residues in human deoxyhypusine synthase. A drastic reduction in enzyme intermediate fo rmation and enzymatic activities was observed with mutant proteins wit h substitution at Lys(287) but, not with those with mutations at resid ues 141, 156, 205, 212, 226, 251, or 338. Lys to Ala or Lys to Arg sub stitution at Lys(329) totally abolished covalent enzyme-substrate inte rmediate formation and deoxyhypusine synthesis activity, indicating th at Lys(329) is the unique site for the enzyme intermediate and that it is absolutely required for deoxyhypusine synthesis in the eukaryotic translation initiation factor 5A precursor. The K329A mutant showed sp ermidine cleavage activity (similar to 6% of the wild type enzyme) sug gesting that in contrast to deoxyhypusine synthesis, spermidine cleava ge can occur without enzyme intermediate formation.